Frackelton Ar

Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.

Constitutively tyrosine phosphorylated p52 Shc in breast cancer cells: correlation with ErbB2 and p66 Shc expression.

Breast cancer cell lines display a wide variety of growth factor receptors, and considerable evidence implicates signaling from these receptors, especially ErbB2, in the important early stages of this disease, contributing to malignant progression. If this is true, then we would hypothesize that a useful prognostic indicator would be the level of activity of a second messenger protein used in common by these receptors. One such second messenger is the Shc adapter protein, which is activated when tyrosine phosphorylated by receptors. Therefore, one prediction from the hypothesis is that the level of tyrosine-phosphorylated Shc (PY-Shc) in breast cancer cell lines would correlate with total receptor tyrosine kinase activity. To begin to test this prediction, we examined Shc tyrosine phosphorylation in a diverse group of breast cancer cell lines that display varied levels of ErbB2. Using Shc immunoprecipitation and anti-phosphotyrosine immunoblotting analysis, we found a strong correlation between the level of ErbB2 overexpression (r=0.91, p < 0.0002) and PY-ErbB2 levels (r=0.89, p=0.0005) compared with the level of tyrosine phosphorylation of the p52 and p46 Shc isoforms. Consistent with Shc tyrosine phosphorylation being driven by ErbB2, an ErbB2-specific tyrosine kinase inhibitor markedly reduced Shc tyrosine phosphorylation. Unexpectedly, although all cell lines had comparable total amounts of p52 and p46 Shc, the amount of an inhibitory Shc isoform, p66, was inversely related to the level of ErbB2 expression (r=− 0.86, p=0.0013). This suggests that reduced p66 Shc expression may play a role in ErbB2-positive breast cancer. In summary, these data are consistent with our prediction that the cellular level of PY-Shc would correlate with the levels of activated ErbB2 displayed by cell lines derived from breast cancers.

Generation of monoclonal antibodies against phosphotyrosine and their use for affinity purification of phosphotyrosine-containing proteins.

Publisher Summary This chapter describes methods for preparing the PY-protein and PYA-protein conjugates, immunizing mice, and screening hybridoma culture fluid for specific antibodies. It compares and contrasts the affinities and fine specificities of these antibodies with those of the earlier anti-ABP monoclonal antibody (2G8) and provides detailed methods for using the most avid and specific monoclonal antibody (1G2) for purifying PY-containing proteins from cells. The first polyclonal and monoclonal antibodies reactive with phosphotyrosine (PY)-containing proteins were generated by immunizing animals with p-aminobenzylphosphonic acid (ABP) diazotized to carrier protein. This analog of phosphotyrosine was chosen over phosphotyrosine as the haptenic group because the phosphonate group, in contrast to the phosphate group of PY, was resistant to enzymatic hydrolysis, and thus might be a more persistent and efficient immunogen. Polyclonal and monoclonal antibodies to ABP, however frequently cross-reacted with a number of other phosphate-containing compounds. One ABP-induced monoclonal antibody, 2G8, had been extremely useful for characterizing and purifying a variety of phosphotyrosyl proteins.

Receptors for platelet-derived growth factor on microvascular endothelial cells

Endothelial cells are widely thought to be unresponsive to platelet-derived growth factor (PDGF) and devoid of PDGF receptors. However, in examining the growth factor responses of microvascular endothelial cells isolated from human omental adipose tissue, we detected PDGF-induced tyrosine phosphorylation of an 180-kD glycoprotein, subsequently identified as the cellular receptor for PDGF by specific immunoprecipitation. Scatchard analysis of 125I-PDGF binding to human microvascular endothelial cells revealed 30,000 PDGF receptors/cell with a kD of 0.14 nM. PDGF stimulated tyrosine phosphorylation of PDGF and other cellular proteins in a dose- and time-dependent manner, with half-maximal receptor phosphorylation occurring at 0.3 nM recombinant human PDGF-BB within 1 min of PDGF exposure. Normal cellular consequences of receptor activation were also observed, including tyrosine phosphorylation of a 42-kD protein and serine phosphorylation of ribosomal protein S6. Furthermore, PDGF was mitogenic for these cells. The finding of functional PDGF receptors on human microvascular endothelial cells suggests an important direct role for PDGF in neovascularization.

Fluorescence changes associated with G-F transformation of actin.

Actin, the major protein component of the thin filaments of striated muscle, can exist either as a monomer, G-actin, of molecular weight 47 000 [l] , or in a polymerized, filamentous form, F-actin. In the thin filaments, actin is associated with tropomyosin in a stoichiometric combination [2] , and with a number of minor proteins which constitute the calcium sensitizing system, troponins and associated proteins [3]. As a prelude to an intensive study of conformational and microenvironmental changes in actin when it is polymerized and then associates with other thin filament proteins and with myosin using fluorescent probes, we undertook a study of the intrinsic fluorescence of actin and the changes which occur upon polymerization.